1. Field of the Invention
This invention relates to new mutant strains of Bradyrhizobium japonicum which in symbiotic combination with leguminous plants increase the nitrogen-fixing capability of the plant. This invention also relates to the commercial practice of inoculating soybeans and other legume seedlings with bacteria to increase effective nodulation and growth of leguminous crops.
2. Description of the Prior Art
The fixation of atmospheric nitrogen associated with specific legumes is the result of a highly specific symbiotic relationship with rhizobial bacteria. These indigenous bacteria dwell in the soil and are responsible for the formation of nodules in the roots of leguminous plants as sites for the nitrogen fixation. Currently, these rhizobia are classified by growth rate in free-living cultures, with the fast-growing organisms being designated as Rhizobium and the slow-growing organisms as Bradyrhizobium. Many of the rhizobial strains are not only host-specific but also differ with respect to capacity for effective symbiosis. Those strains which are able to infect a plurality of host plants across species or genus lines are said to be "cross-nodulating." Commercial inocula generally consist of a mixture of rhizobial strains to insure the widest potential for effective symbiosis within the appropriate crop.
In the art of soybean cultivation, unspecified strains of B. japonicum have become recognized as standard inoculants for initiating nodulation and nitrogen fixation.
The symbiotic properties of Bradyrhizobium japonicum mutants with altered tryptophan biosynthetic pathways have been investigated. Wells and Kuykendall [J. Bacteriol. 156: 1356-1358 (1983)] isolated a series of tryptophan-requiring mutants of B. japonicum I-110ARS. These mutants fell into five classes according to their enzyme deficiencies. Only mutants that were defective in tryptophan synthetase, the last step of the tryptophan biosynthetic pathway, were capable of forming nodules. Other mutants, with defects earlier in the pathway, did not nodulate as auxotrophs but did as prototrophic revertants. Also, B. japonicum I-110 mutants that are resistant to 5-methyltryptophan and that constitutively overproduce tryptophan and tryptophan pathway products have been isolated and studied [Hunter, Appl. Environ Microbiol. 53: 1051-1055 (1987)]. Generally, these bacteria were poor nodulators and poor nitrogen fixers.
Kaneshiro and Kwolek [Plant Sci. 42: 141-146 (1985)] reported that trypotophan catabolic variants of a Bradyrhizobium sp. (soybean) strain L-259 had improved symbiotic properties. It is characteristic of these catabolic mutants, when grown in the presence of tryptophan, to degrade tryptophan rapidly, to accumulate large amounts of indole compounds, and to produce a tan pigment.